Maëlle Saunier

Human papillomavirus DNA prevalence and type distribution in anal carcinomas worldwide

Knowledge about human papillomaviruses (HPV) types involved in anal cancers in some world regions is scanty. Here, we describe the HPV DNA prevalence and type distribution in a series of invasive anal cancers and anal intraepithelial neoplasias (AIN) grades 2/3 from 24 countries. We analyzed 43 AIN 2/3 cases and 496 anal cancers diagnosed from 1986 to 2011. After histopathological evaluation of formalin‐fixed paraffin‐embedded samples, HPV DNA detection and genotyping was performed using SPF‐10/DEIA/LiPA25 system (version 1). A subset of 116 cancers was further tested for p16INK4a expression, a cellular surrogate marker for HPV‐associated transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance in the anal cancer data set. HPV DNA was detected in 88.3% of anal cancers (95% confidence interval [CI]: 85.1–91.0%) and in 95.3% of AIN 2/3 (95% CI: 84.2–99.4%). Among cancers, the highest prevalence was observed in warty–basaloid subtype of squamous cell carcinomas, in younger patients and in North American geographical region. There were no statistically significant differences in prevalence by gender. HPV16 was the most frequent HPV type detected in both cancers (80.7%) and AIN 2/3 lesions (75.4%). HPV18 was the second most common type in invasive cancers (3.6%). p16INK4a overexpression was found in 95% of HPV DNA‐positive anal cancers. In view of the results of HPV DNA and high proportion of p16INK4a overexpression, infection by HPV is most likely to be a necessary cause for anal cancers in both men and women. The large contribution of HPV16 reinforces the potential impact of HPV vaccines in the prevention of these lesions.

Human Papillomavirus Genotype Distribution in External Acuminata Condylomata: A Large French National Study (EDiTH IV)

BACKGROUND External acuminata condylomata (EAC) are among the most common sexually transmitted diseases. Although it is understood that low-risk human papillomavirus (HPV) genotypes 6 and 11 are associated with EAC, there have only been a few, small, published studies reporting the genotype-specific prevalence of HPV. The objective of our study was to assess the prevalence of HPV genotypes for a large number of cases involving both men and women and to evaluate the potential benefit of a quadrivalent (genotypes 6, 11, 16, and 18) HPV vaccine in France. METHODS A total of 256 women and 260 men who presented with EAC to French gynecologists, dermatologists, and proctologists were prospectively recruited during the period January through April 2007. Specimens were collected with a cytobrush, and the HPV genotype was determined using the INNO-LiPA assay (Innogenetics), which detects 24 HPV genotypes. RESULTS Four hundred twenty-three beta-globin-positive samples could be analyzed. The median age of patients was 30 years (range, 18-72 years). The overall prevalence of HPV DNA in patients with EAC was 99% (33% of patients were coinfected with another pathogen). Low-risk genotypes predominated, with a prevalence of 89%. The most prevalent genotypes were 6 (69%) and 11 (16%), followed by 16 (9%), 51 (8%), 52 (7%), 66 (6%) 53 (5%), 31 (3%), and 18 (3%). The cumulative prevalence of genotypes 6 and 11 was 83%, and the cumulative prevalence of genotypes 6, 11, 16, and 18 was 88%. CONCLUSIONS This study is, to our knowledge, the first large, multicenter survey to provide solid data on HPV genotype distribution among patients with EAC. Our results provide strong evidence that, in France, the most prevalent HPV genotypes in persons with EAC are 6 and 11. Because of its 99% efficacy for the prevention of EAC and a vaccine coverage of 100%, the quadrivalent HPV vaccine could prevent 62%-87% of EAC cases in France.

Analysis of Human Papillomavirus Type 16 (HPV16) DNA Load and Physical State for Identification of HPV16-Infected Women with High-Grade Lesions or Cervical Carcinoma

ABSTRACT Integration of human papillomavirus (HPV) DNA into the host cell genome is a frequent event in cervical carcinogenesis, even though this phenomenon does not seem to be mandatory for cervical cancer development. Our objective was to describe the load and physical state of HPV type 16 (HPV16) DNA in a series of cervical samples representative of the natural history of cervical cancer. We used a combination of three real-time PCR assays targeting E6, E2, and albumin genes to calculate HPV16 load (E6 and albumin) and the E2/E6 ratio as a surrogate of integration. This method was applied to 173 HPV16-positive cervical samples. Results show that viral load increases with the lesion grade (from 102 HPV16 DNA copies per 103 cells in normal samples up to 56,354 copies per 103 cells in cancers), while E2/E6 ratio decreases (from 1 in normal samples down to 0.36 in cancers). We propose that, according to this technique, an HPV16 viral load of higher than 22,000 copies/103 cells or an E2/E6 ratio of lower than 0.50 allows the identification of women with prevalent high-grade lesions or worse with a high specificity. In conclusion, both viral load and E2/E6 ratio, used in combination with an appropriate cutoff value, are suitable to screen women with prevalent cervical intraepithelial neoplasia grade 2 or 3 or cancer. Therefore, these assays would be useful in addition to routine HPV testing to more accurately identify women with (pre)cancerous lesions.

Large contribution of human papillomavirus in vaginal neoplastic lesions: A worldwide study in 597 samples

AIM This work describes the human papillomavirus (HPV) prevalence and the HPV type distribution in a large series of vaginal intraepithelial neoplasia (VAIN) grades 2/3 and vaginal cancer worldwide. METHODS We analysed 189 VAIN 2/3 and 408 invasive vaginal cancer cases collected from 31 countries from 1986 to 2011. After histopathological evaluation of sectioned formalin-fixed paraffin-embedded samples, HPV DNA detection and typing was performed using the SPF-10/DNA enzyme immunoassay (DEIA)/LiPA25 system (version 1). A subset of 146 vaginal cancers was tested for p16(INK4a) expression, a cellular surrogate marker for HPV transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance. RESULTS HPV DNA was detected in 74% (95% confidence interval (CI): 70-78%) of invasive cancers and in 96% (95% CI: 92-98%) of VAIN 2/3. Among cancers, the highest detection rates were observed in warty-basaloid subtype of squamous cell carcinomas, and in younger ages. Concerning the type-specific distribution, HPV16 was the most frequently type detected in both precancerous and cancerous lesions (59%). p16(INK4a) overexpression was found in 87% of HPV DNA positive vaginal cancer cases. CONCLUSIONS HPV was identified in a large proportion of invasive vaginal cancers and in almost all VAIN 2/3. HPV16 was the most common type detected. A large impact in the reduction of the burden of vaginal neoplastic lesions is expected among vaccinated cohorts.

HPV prevalence, viral load and physical state of HPV‐16 in cervical smears of patients with different grades of CIN

Human papillomavirus (HPV) infection is the most important event in malignant transformation of human cervical epithelium. We analysed in cervical smears, HPV genotypes with a focus on single/multiple infections, then characteristics of HPV‐16 infections (presence of other genotypes, viral load and physical state) according to the grade of histological lesions. The purpose of this study was to know if these parameters could allow to differentiate histological diagnoses. DNA was extracted from 363 cervical samples corresponding to 24 cases without lesion, 96 CIN1, 92 CIN2, 144 CIN3 and 7 cancers. Our results show that HPV‐16 was predominant and its prevalence increased with the severity of lesions (CIN1: 27.1%; CIN3: 65.3%). In addition, we showed that the frequency of single infections, as compared with multiple infections, increased with the severity of the lesion (CIN1: 25.0%; CIN3: 54.8%). Among HPV‐16 positive samples (n = 170), we found that viral load, determined on cervical samples by real‐time PCR, did not vary significantly according to the different CIN grades. Concerning HPV‐16 integration, the mixed and integrated HPV‐16 forms, already present in women with normal histology, increased to the benefit of pure episomal forms with the severity of lesions (normal cervix: 28.6%; CIN3: 73.8%). Thus, our data raise the question of the viral load as a valuable clinical parameter to discriminate between lesion grades. Moreover, we emphasize integration as an early event in cervical carcinogenesis, increasing with the severity of lesions. Finally, this study underlines the importance of single versus multiple infections linked to the severity of CIN.

Human papillomavirus genotype distribution in low-grade squamous intraepithelial lesions in France and comparison with CIN2/3 and invasive cervical cancer: The EDiTH III study

OBJECTIVES In the present study (EDiTH III study), the genotype-specific prevalence of HPV in low-grade squamous intraepithelial lesions (LSIL) was estimated to predict the potential benefit of HPV vaccination in France. This prevalence was compared to that previously reported in France in high-grade cervical intraepithelial neoplasia (CIN2/3, EDiTH II study) and squamous cell carcinoma (SCC, EDiTH I study) to identify the genotypes preferentially associated with a progression to malignancy. METHODS 397 smears with LSIL diagnosis (Preservcyt) were retrospectively collected in different centres in France and genotyped using the INNO-LiPA assay allowing the detection of 24 HPV genotypes. RESULTS HPV was found in 98% of cases. The most prevalent genotypes in LSIL in France were HPV 66 (25%), HPV 16 (21%), HPV 53 (18%), 51 (17%) and 52 (14%). HPV 16 and/or 18 were present in 28% and HPV 6, 11, 16 and/or 18 in 33% of LSIL. The highest SCC/LSIL prevalence ratios were shown for HPV 16, 33 and 18. CONCLUSIONS With a 95% vaccine efficacy on CIN1 and theoretical vaccine coverage of 100%, HPV vaccination might prevent 27% (with a 16, 18 bivalent vaccine) and up to 32% (with a 6, 11, 16, 18 quadrivalent vaccine) of LSIL cases in France. In this study, LSIL related to HPV 16, 18 or 33 are at highest risk of progression to malignancy and thus could require a stringent surveillance. Conversely, anxiety and over-treatment could be avoided in women with low risk of progression.

Apoptotic HPV Positive Cancer Cells Exhibit Transforming Properties

Previous studies have shown that DNA can be transferred from dying engineered cells to neighboring cells through the phagocytosis of apoptotic bodies, which leads to cellular transformation. Here, we provide evidence of an uptake of apoptotic-derived cervical cancer cells by human mesenchymal cells. Interestingly, HeLa (HPV 18+) or Ca Ski (HPV16+) cells, harboring integrated high-risk HPV DNA but not C-33 A cells (HPV-), were able to transform the recipient cells. Human primary fibroblasts engulfed the apoptotic bodies effectively within 30 minutes after co-cultivation. This mechanism is active and involves the actin cytoskeleton. In situ hybridization of transformed fibroblasts revealed the presence of HPV DNA in the nucleus of a subset of phagocytosing cells. These cells expressed the HPV16/18 E6 gene, which contributes to the disruption of the p53/p21 pathway, and the cells exhibited a tumorigenic phenotype, including an increased proliferation rate, polyploidy and anchorage independence growth. Such horizontal transfer of viral oncogenes to surrounding cells that lack receptors for HPV could facilitate the persistence of the virus, the main risk factor for cervical cancer development. This process might contribute to HPV-associated disease progression in vivo.

Disagreement in high-grade/low-grade intraepithelial neoplasia and high-risk/low-risk HPV infection: clinical implications for anal cancer precursor lesions in HIV-positive and HIV-negative MSM

Anal condylomata are common in HIV-positive individuals and among men who have sex with men (MSM). Generally attributable to infection by low-risk human papillomaviruses (HPVs), condylomata are considered benign low-grade squamous intraepithelial lesions (SILs). However, anal condylomata have occasionally been linked to high-grade SIL and to oncogenic, high-risk HPVs. Here we describe the range of intraepithelial lesions and of the associated HPVs in heterosexual men and women and MSM. Perianal and anal condylomata were collected from 243 patients (56 heterosexual women, 61 heterosexual men and 126 MSM, including 41 HIV-positive MSM). We assessed lesion histology and HPV genotype. Prevalence estimates and Poisson models were used. Irrespective of HIV infection status, MSM showed a higher proportion of condylomata as high-grade SILs compared to heterosexual men/women. High-grade SILs were also more prevalent in anal than in perianal lesions in all patient groups. HIV-positive MSM exhibited increased prevalence ratio (4.6; 95% confidence interval 2.1-10.0) of perianal low-grade SILs containing only high-risk HPVs compared to HIV-negative MSM. In addition, more than 64% of anal SILs with a high-grade component, regardless of HIV infection, were exclusively associated with low-risk HPVs. In anal condylomata, both high-grade and low-grade SILs can be associated with high-risk and/or low-risk HPVs. Particularly, low-grade perianal SILs associated with high-risk HPVs were common in HIV-positive MSM, while presence of only low-risk HPVs in high-grade SILs were common in both MSM groups. Our findings sound a note of caution for the common clinical practice for the treatment of anal condylomata as benign lesions in MSM and HIV-positive patients.

HPV DNA prevalence and type distribution in anal carcinomas worldwide

Knowledge about human papillomaviruses (HPV) types involved in anal cancers in some world regions is scanty. Here, we describe the HPV DNA prevalence and type distribution in a series of invasive anal cancers and anal intraepithelial neoplasias (AIN) grades 2/3 from 24 countries. We analyzed 43 AIN 2/3 cases and 496 anal cancers diagnosed from 1986 to 2011. After histopathological evaluation of formalin‐fixed paraffin‐embedded samples, HPV DNA detection and genotyping was performed using SPF‐10/DEIA/LiPA25 system (version 1). A subset of 116 cancers was further tested for p16INK4a expression, a cellular surrogate marker for HPV‐associated transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance in the anal cancer data set. HPV DNA was detected in 88.3% of anal cancers (95% confidence interval [CI]: 85.1–91.0%) and in 95.3% of AIN 2/3 (95% CI: 84.2–99.4%). Among cancers, the highest prevalence was observed in warty–basaloid subtype of squamous cell carcinomas, in younger patients and in North American geographical region. There were no statistically significant differences in prevalence by gender. HPV16 was the most frequent HPV type detected in both cancers (80.7%) and AIN 2/3 lesions (75.4%). HPV18 was the second most common type in invasive cancers (3.6%). p16INK4a overexpression was found in 95% of HPV DNA‐positive anal cancers. In view of the results of HPV DNA and high proportion of p16INK4a overexpression, infection by HPV is most likely to be a necessary cause for anal cancers in both men and women. The large contribution of HPV16 reinforces the potential impact of HPV vaccines in the prevention of these lesions.

Real-time duplex PCR for simultaneous HPV 16 and HPV 18 DNA quantitation ☆

HPV 16 and HPV 18 are responsible for more than 75% of cervical cancers and high HPV 16 loads are associated with both prevalent and incident lesions. The objective of the present study was to develop a method allowing the detection and quantitation of HPV 16 and 18 DNA to improve future strategies for cervical cancer screening. A duplex real-time PCR allowing the simultaneous quantitation of both HPV 16 and HPV 18 was carried out. Mixes of HPV 16 and HPV 18 whole genome plasmids were prepared to test a wide range of viral DNA concentrations. The values obtained for each mix of plasmids with the simplex and the duplex PCR were very close to the theoretical values except when a HPV type represented only 1:1000 genome equivalent or lower than the concurrent type. Cervical samples harboring HPV 16, HPV 18 or both types were tested by comparing the results with simplex and duplex real-time PCR assays. HPV 16 and HPV 18 genome titers were similar with the two assays. In conclusion, the real-time duplex PCR proved to be robust for HPV 16 and HPV 18 DNA quantitation.